We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use a lateral flow device to detect amplification products. Such devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several "moderate complexity" and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 200 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). Preliminary studies suggest a limit of detection of 100 copies/mL plasma when nucleic acids extracted from plasma samples with a sequence-specific capture method are used as templates for RT-HDA. We propose to further test the specificity and sensitivity of the HIV test with clinical specimens obtained from 5 high-risk patient recruitment sites. Assays will be performed at BioHelix, and at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. In addition, we propose to perform a validation study at the National Institute for Communicable Diseases (NICD) of South Africa to gain clearance to sell kits in South Africa. Finally, we may also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. By the end of Phase II, we will have preliminary data for submission of an IDE to the FDA to kick-off a clinical study to seek pre market approval (PMA) for sale of the assay system for human diagnostics in the US. PUBLIC HEALTH RELEVANCE: More than 90 % of the 40 million human immunodeficiency virus (HIV)-infected persons live in Third World countries (see www.unaids.org/bangkok2004/GAR2004_html/GAR2004_03_en.htm). Unfortunately, performing HIV molecular tests in resource poor settings is difficult. BioHelix's proprietary helicase dependent amplification (HDA) platform is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. HDA is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). Indeed, HDA can be performed in a simple $100 water bath. To illustrate the potential of this technology for the third world, we currently sell HDA reagents as part of a teaching kit for use in high schools (sold through Carolina Biological Supply). BioHelix is combining this HDA technology with a low cost (~$2- $3.75) disposable device specifically designed to perform molecular tests without contaminating the laboratory with amplicons. The device uses a lateral flow system to detect amplification products. Lateral flow devices are common in many moderately complex, and CLIA-waived tests listed on the Food and Drug Administration (FDA) device database. We believe this combination of characteristics makes our proposed product ideal for the HIV vaccine trial sites. A low cost nucleic acid screening test will help identify those people and reduce the spread of the HIV epidemic as well as benefit participants in HIV vaccination trials who become infected with the virus. An estimated 16 to 22 million persons aged 18-64 years are tested annually for HIV in the United States (see http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5347a4.htm). Most of the near patient testing relies on rapid tests like OraQuick while most hospital screening programs use enzyme immuno assay (EIA). Walensky et al. (2008) recently reported that using western blot alone as a confirmation test for oral rapid tests provides conclusive HIV status in only 50% of patients at first follow-up. Such patients are typically asked to return for testing in ~ 1 month. In contrast, performing an HIV-1 RNA test to confirm the rapid test, when a western blot fails to do so, improves this rate to 96.2% in the first round of testing. Considering that our assay system relies of minimal instrumentation we feel our test can offer an additional layer of confirmation for OraQuick positives at the near patient setting and in the developing world and thus relieve the need for asking patients to return for testing in 1 month.